research highlights

BIOCHEMISTRY CRISPR snapshots of a gene-editing tool

From single-molecule functional studies complexes on tethered DNA substrates by to atomic-resolution structures, a windfall total-internal-reflection fluorescence micros- of data sheds light on the Cas9 mechanism copy (Sternberg et al., 2014). Using unlabeled of targeted DNA scission. substrate competitors, the researchers were An inexhaustible source of research tools able to probe binding kinetics and homology for genetic trickery, bacteria have recently requirements for target recognition. delivered another revolutionary technology. Both DNA binding and cleavage were The Cas9 endonuclease, which in nature uses shown to require the recognition of a tri- short guide RNAs to protect bacteria against nucleotide sequence (protospacer-adjacent genome invaders, has been successfully har- motif, or PAM) that is not part of but is adja- nessed for on-demand DNA targeting in cent to the RNA-targeted sequence. Whereas multiple cell types and organisms. Now three Cas9-RNA bound to PAM-enriched regions separate studies take a closer look at Cas9 even in the absence of target sequences, it structure and function. did not detect even perfectly homologous For Jennifer Doudna, a biochemist targets without an intact PAM. DNA melt- Structural and single-molecule fluorescence studies and RNA-interference aficionado at the ing and RNA-DNA hybridization were provide insight into the Cas9 mechanism. Figure University of California (UC), Berkeley, it all shown to originate at the PAM and then reproduced with permission from J. Doudna, D. Taylor and S. Sternberg. started in the early 2000s with a basic scien- spread towards the distal end of the target. tific question: could bacterial clustered, regu- And although this master role for PAMs was larly interspaced, short palindromic repeats unexpected, Doudna hopes that scientists authors turned to single-particle electron (CRISPR) and their associated (cas) genes can use the findings to minimize off-target microscopy. The three-dimensional structure represent an RNA interference–like immune effects by monitoring PAM distribution reconstructions showed that nucleic acid– pathway? and PAM-adjacent sequences in engineered bound Cas9 exhibited a massive conforma-

Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature “We certainly did not set out to discover genomes. tional change that repositions the two lobes a genome engineering technology,” says In a separate study, Doudna’s group along to clasp around the target sequence. Such a Doudna. But when she and Emmanuelle with that of her UC Berkeley colleague Eva conformational rearrangement was con- Charpentier at the Helmholtz Center for Nogales set out to reveal the structural basis firmed by an independent report from the npg Infection Research later realized that Cas9 for Cas9 function (Jinek et al., 2014). They groups of Feng Zheng at the Broad Institute not only provides adaptive immunity in solved crystal structures of the apo forms of and Osamu Nureki at the University of Tokyo many bacteria but can also be reprogrammed two different Cas9 enzymes, each represent- that presented the crystal structure of a Cas9- by a single RNA to target virtually any DNA ing a major Cas9 subtype. The structures RNA-DNA complex (Nishimasu et al., 2014). sequence, the potential for genome engineer- revealed bi-lobed enzyme architecture, with “What we really now want to understand is ing was too blatant to ignore. Unlike zinc- a common catalytic core hosted in one of the whether this large conformational change is finger nucleases and transcription activator– lobes and a divergent α-helical domain in actually part of the mechanism to open the like effector nucleases, “this system utilizes a the other. The latter is substantially smaller double-stranded DNA duplex,” says Doudna, single protein whose specificity is changed by in one of the Cas9 enzymes, and this evolu- explaining that the enzyme has no intrinsic simply altering the guide RNA, and it is very tionary variability could be further exploited or coupled ATPase activity. Hopefully further amenable to targeting multiple sequences in to engineer more compact Cas9 variants or mechanistic snapshots will follow. the same cell,” explains Doudna. to insert additional functionalities, explains Petya V Krasteva Despite the simplicity and broad adop- Doudna. The authors identified the putative RESEARCH PAPERS tion of the technology, however, the molec- nucleic acid binding clefts and PAM recogni- Jinek, M. et al. Structures of Cas9 endonucleases reveal ular mechanisms of Cas9 function have tion motifs, as well as the structural require- RNA-mediated conformational activation. Science remained enigmatic. To study the enzyme ments for PAM sequence specificity. doi:10.1126/science.1247997 (6 February 2014). in action, Doudna’s group joined forces with Interestingly, in both structures the Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, that of Eric Greene at Columbia University. enzymes appeared autoinhibited, with cata- 935–949 (2014). The team generated a quantum dot–labeled lytic residues facing away from the target- Sternberg, S.H. et al. DNA interrogation by the CRISPR Cas9 variant and used a DNA-curtain assay binding clefts. To capture the conformation RNA-guided endonuclease Cas9. Nature 507, 62–67 to monitor the action of single Cas9-RNA of the guide- and target-bound forms, the (2014).

nature methods | VOL.11 NO.4 | APRIL 2014 | 365